Cigarette smoke impairs the hematopoietic supportive property of mesenchymal stem cells via the production of reactive oxygen species and NLRP3 activation

Mesenchymal stem cells (MSCs) play important roles in tissue homeostasis by providing a supportive microenvironmental niche for the hematopoietic system. Cigarette smoking induces systemic abnormalities, including an impeded recovery process after hematopoietic stem cell transplantation. However, the role of cigarette smoking-mediated alterations in MSC niche function have not been investigated. In the present study, we asked whether exposure to cigarette smoking extract (CSE) disrupts the hematopoietic niche function of MSCs, and pathways impacted. To investigate the effects on bone marrow (BM)-derived MSCs and support of hematopoietic stem and progenitor cells (HSPCs), we examined the impact of 3R4F as a reference CSE, and HSPC-supporting function was determined. Both direct ex vivo and systemic in vivo MSC exposure to 3R4F resulted in impaired engraftment in a humanized mouse model. Furthermore, transcriptomic profile analysis showed significantly upregulated signaling pathways related to reactive oxygen species (ROS), inflammation, and aging in 3R4F-treated MSCs. 3R4F-exposed MSCs also developed impaired HSPC-supportive function, as confirmed by colony forming unit (CFU) assays and HSPC transplantation into NSG mice, and were further restored by the ROS inhibitor N-acetyl-l-cysteine (NAC). Finally, ingenuity pathway analysis revealed the activation of NLRP3 inflammasome signaling pathway in 3R4F-treated MSCs, and pretreatment with the NLRP3 inhibitor MCC950 rescued the HSPC-supporting ability of 3R4F-treated MSCs. In conclusion, these findings indicate that exposure to CSE reduces the HSPC-supportive function of MSCs by inducing robust ROS production and subsequent NLRP3 activation. RNA sequencing (RNA-seq) was performed by Theragen Bio (Seongnam, South Korea) using Illumina technology as previously described, with modifications (22). Total RNA was extracted and purified from hMSCs incubated with or without 5% 3R4F using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Libraries were generated using the Illumina TruSeq strand mRNA sample preparation kit (Illumina, San Diego, CA, USA) and sequenced on a NovaSeq 6000 (2 x 150 paired end sequencing, Illumina) according to the manufacturer's protocol.